Modified oligonucleotides, their preparation and their use

• US 6,987,177 B2
• Filed: 08/19/2002
• Issued: 01/17/2006
• Est. Priority Date: 11/04/1994
• Status: Expired due to Fees

• Current Assignee: Sanofi-Aventis Deutschland GmbH (Sanofi )
• Original Assignee: Aventis Pharma Deutschland GmbH (Sanofi )
• Inventors: Seela, Frank, Thomas, Horst
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/222,825
• Publication Number:
  US 20030096981A1
• Time in Patent Office: 1,247 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.5, 536/23.5, 536/27.2, 435/6, 435/91.1
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/044 Pyrrole radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

Modified oligonucleotides which possess at least one substituted 7-deazapurine base form more stable hybridization complexes with nucleic acids than unsubstituted analogs. They are useful as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents. Processes for preparing them are provided.

Claims

1. A nucleotide monomer of the formula V

in which

V is oxy, sulfanediyl or imino;

Y^b is oxy, sulfanediyl or methylene;

a is oxy, sulfanediyl or methylene;

R^2b is hydrogen, OR¹², C₁-C₆-alkenyloxy, in particular allyloxy, halogen, azido or NR¹⁰R¹¹;

R¹ is a protective group which is customary in nucleotide chemistry;

R^1b is a linker, optionally linked to a solid support, or is a radical of the formula IIIc or IIId

in which U is (C₁-C₁₈)-alkoxy, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, O—

R⁷, S—

R⁷ or a radical of the formula IV

(OCH₂CH₂)_pO(CH₂)_qCH₂R⁵ 

 

(IV)

in which R⁵ is H;

Q is a radical —

NR⁸R⁹,

R⁷ is —

(CH₂)₂—

CN;

R⁸ and R⁹ are identical or different and are C₁-C₆-alkyl, or, together with the nitrogen atom carrying them, are a 5-9-membered heterocyclic ring which can additionally contain a further O, S or N heteroatom;

E′ and

F′

are, independently of each other, H, OR¹² or NR¹⁰R¹¹,

R¹⁰ and R¹¹ are identical or different and are hydrogen or an amino protective group which is customary in nucleotide chemistry, or R¹⁰ and R¹¹ together form an amino protective group which is customary in nucleotide chemistry,

R¹² is hydrogen or a hydroxyl protective group which is customary in nucleotide chemistry;

R¹⁵ and R¹⁶ are, independently of each other,

(1) hydrogen,

(2) halogen,

(3) (C₁-C₁₀)-alkyl,

(4) (C₂-C₁₀)-alkenyl,

(5) (C₂-C₁₀)-alkynyl,

(6) NO₂,

(7) NH₂,

(8) cyano,

(9) —

S—

(C₁-C₆)-alkyl,

(10) (C₁-C₆)-alkoxy,

(11) (C₆-C₂₀)-aryloxy,

(12) SiH₃

(14) a radical as described under (3), (4) or (5) which is substituted by one or more radicals selected from the group consisting of SH, S—

(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —

NR(c)R(d), —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(g), —

NR(e)R(f), and —

NR(e)R(g), or by a polyalkyleneglycol radical of the formula —

[O—

(CH₂)_r]_s—

NR(c)R(d), where r and s are, independently of each other an integer from 1 to 18, wherein any of the foregoing OH, SH, —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(d), —

NR(e)R(f), and —

NR(e)R(g) or —

NR(c)R(g) groups optionally carries a protective group which is customary in nucleotide chemistry or is linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve for labeling a DNA or RNA probe, or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or

(15) is a radical as defined under 3, 4 or 5 in which from one to all the H atoms are substituted by halogen;

R(a) is OH, (C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T,

where T is an alkylcarboxyl group or alkylamino group

which is linked, optionally via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,

R(b) is hydroxyl, (C₁-C₆)-alkoxy or —

NR(c)R(d),

R(c) and R(d) are, independently of each other, H or (C₁-C₆)-alkyl which is unsubstituted or substituted by —

NR(e)R(f) or —

NR(e)R(g),

R(e) and R(f) are, independently of each other, H or (C₁-C₆)-alkyl,

R(g) is (C₁-C₆)-alkyl-COOH;

with the proviso that when R¹⁵ and R¹⁶ are identical, they cannot be hydrogen, NO₂, NH₂, cyano or SiH₃;

wherein functional groups are optionally protected with a protective group which is customary in nucleotide chemistry, and the curved bracket indicating that R^2b and the adjacent —

Y^bR^2b radical can be located in the 2′ and

3′

position or else, conversely, in the 3′ and

2′

position.

2. The nucleotide monomer of the formula V of claim 1, wherein

R¹⁵ is (1) NO₂,

(2) NH₂,

(3) —

S—

(C₁-C₆)-alkyl,

(4) (C₁-C₆)-alkoxy,

(5) (C₆-C₂₀)-aryloxy,

(6) SiH₃,

(8) (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl, any of which may be substituted by one or more radicals selected from the group consisting of SH, S—

(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —

NR(c)R(d), —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(g),

—

NR(e)R(f) and —

NR(e)R(g), or by a polyalkylene glycol radical of the formula —

[O—

(CH₂)_r]_s—

NR(c)R(d), where r and a are, independently of each other, an integer between 1 and 18, wherein any of the foregoing OH, SH, —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(d), —

NR(e)R(f), —

NR(e)R(g) or —

NR(c)R(g) groups optionally is linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or

(9) is (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl in which from one to all the H atoms are substituted by halogen; and

R¹⁶ is hydrogen.

3. The nucleotide monomer of formula V of claim 1, wherein R^1b is a succinyl linker, linked to an amino-functionalized or methylamino-functionalized solid support by any amide or methylamide bond.

4. The nucleotide monomer of formula V of claim 1, wherein at least one of R⁸ and R⁹ is isopropyl or ethyl.

7. The nuclectide monomer of formula V of claim 1, wherein R^1b is a succdinyl linker radical, bearing an activated ester.

8. The nucleotide monomer of formula V of claim 1, wherein at least one of R⁸ and R⁹ is

5. A compound of the formula VI

in which, independently of each other, U′

=U″

=U′

″

is hydroxyl or mercapto;

e and f are 0 or 1;

R¹³ is hydrogen, OH, C₁-C₁₈-alkoxy, or C₁-C₆-alkenyloxy;

E and F are, independently of each other, H, OH or NH_2;

and

R¹⁵ and R¹⁶ are, independently of each other,

(1) hydrogen,

(2) halogen,

(3) (C₁-C₁₀)-alkyl,

(4) (C₂-C₁₀)-alkenyl,

(5) (C₂-C₁₀)-alkynyl,

(6) NO₂,

(7) NH₂,

(8) cyano,

(9) —

S—

(C₁-C₆)-alkyl,

(10) (C₁-C₆)-alkoxy,

(11) (C₆-C₂₀)-aryloxy,

(12) SiH₃,

(14) a radical as described under (3), (4) or (5) which is substituted by one or more radicals selected from the group consisting of SH, S—

(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —

NR(c)R(d), —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(g), —

NR(e)R(f), and —

NR(e)R(g), or by a polyalkyleneglycol radical of the formula —

[O—

(CH₂)_r]_s—

NR(c)R(d), where r and s are, independently of each other an integer between 1 to 18, wherein any of the foregoing OH, SH, —

CO—

R(b), —

NH—

CO—

NR(c)R(d), —

NR(c)R(d), —

NR(e)R(f), and —

NR(e)R(g) or —

NR(c)R(g) groups optionally is linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or are a radical as defined under (3), (4) or (5) in which from one to all the H atoms are substituted by halogen;

R(a) is OH, (C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T, with T representing an alkylcarboxyl or alkylamino group which is linked, where appropriate via a further linker, to one or more groups which favor intracellular uptake, or serve as labeling for a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving,

R(b) is hydroxyl, (C₁-C₆)-alkoxy or —

NR(c)R(d),

R(c) and R(d) are, independently of each other, H or (C₁-C₆)-alkyl which is unsubstituted or substituted by —

NR(e)R(f) or —

NR(c)R(g)

R(e) and R(f) are, independently of each other, H or (C₁-C₆)-alkyl,

R(g) is (C₁-C₆)-alkyl-COOH,

with the proviso that when R¹⁵ and R¹⁶ are identical, they cannot be hydrogen, NO₂, NH₂, cyano or SiH₃;

with compounds of the formula VI being excepted in which R¹⁶ is H and R¹⁵ is

(C₂-C₁₀)-alkynyl which is substituted by —

NR(c)R(d) or —

NR(e)R(f); and

with the additional proviso that e and f are not O if E is OH or NH₂, and F is OH, R¹⁶ is hydrogen and R¹⁵ is Br, Cl, F, cyano, (C₁-C₄)-alkyl, (C₂-C₄)-alkenyl or (C₂-C₄) -alkynyl.

6. The compound of formula VI of claim 5, wherein at least one of R¹⁵ and R¹⁶ is a radical defined under (3), (4), or (5) in which from one to all of the H atoms is/are substituted by halogen, and said halogen is fluorine.

9. A polymerase chain reaction, wherein nucleotide triphosphates polymerize and hybridize to template DNA to amplify desired sequences, and wherein one or more purine nucleotide triphosphates are replaced by one or more of the 7-deazapurine nucleotides of formula VI of claim 5, wherein e=f=1;

and R¹³ is OH.

10. The reaction of claim 9, wherein R¹⁶ is H;

and R¹⁸ is halogen.

11. A method of sequencing polynucleotides, wherein modified purine nucleotide triphosphates are used as chain terminators or chain labeling agents, and wherein one or more purine nucleotide triphosphates are replaced by one or more of the 7-deazapurine nucleotides of formula VI of claim 5, wherein e=f=1;

and R¹³ is H or OH.